Immobilization of linamarase on non-porous glass beads

Process Biochemi~'tO' Vol. 33, No. 5, pp. 491-494, 199g ~ 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0032-9592/98 $19.00 + 0.00 ELSEVIER P ! 1:

S0032-9592197)00093-9

Immobilization of linamarase on non-porous glass beads C. O. I k e d i o b i , "* M. S t e v e n s b a n d L. L a t i n w o ~ ~'Department of Chemistry, College of Arts and Sciences, Florida A&M University, Tallahassee. FL 32307-3700, USA hDiagnostic Division, Abbott Laboratories, Chicago, IL 60064, USA ~Department of Biology, College of Arts and Sciences, Florida A&M University, Tallahassee, FL 32307-3700, USA (Received 18 September 1997; accepted 19 October 1997)

Abstract

The immobilization of purified linamarase [/~-D-glucohydrolase, EC:3.2.1.21] onto non-porous glass beads involved the silanization of tile HF-treated glass beads with 2% 7-aminopropyl-triethoxysilane in acetone, covalent coupling of the alkyl amine to glutaraldehyde and subsequent attachment of the enzyme molecule to glutaraldehyde via a SchiWs base linkage. The immobilized linamarase catalyzed the hydrolysis of its natural substrate, linamarin (2-hydroxyisobutyro-nitrile-/]-D-glucopyranoside) and the synthetic substrate analog, p-nitrophenyl-/~-D-glucopyranoside (pNP-/~-D-glucopyranoside). Glucono-l,5-1actone inhibited the immobilized enzyme competitively irrespective of which of the two substrates was used, while imidazole showed competitive inhibition with linamarin as substrate but non-competitive inhibition with pNP-/~-Dglucopyranoside. The K~ values obtained for glucono-l,5-1actone were 2.1t4 mM and 0.97 mM with linamarin and pNP-/~-o-glucopyranoside as substrates, respectively. The K, values for imidazole as inhibitor were 6.00 mM and 38.20 mM with these two substrates, respectively. The energies of activation, E,,, for the reaction catalyzed by the immobilized enzyme with linamarin and pNP-/~-D-glucopyranoside as substrates were 4.00 kcal/mol and 5.7kcal/mol, respectively. Determination of the operational stability of the immobilized enzyme gave a half-life of 14 days at 27°C assuming continual use of the immobilized enzyme in a fixed-bed reactor for the hydrolysis of cyanogenic glycosides. © 1998 Elsevier Science Ltd. All rights reserved

Keywords:linamarase, immobilization, non-porous glass beads, kinetic behavior.

glucosides, which yields, among other products, HCN, a potent inhibitor of cytochrome oxidase, and other respiratory enzymes [1]. In plant tissues, the enzyme and its natural substrates (linamarin and lotaustralin) are localized in different subcellular organelles. However, during mechanical injury or the intentional maceration of the tissues associated with processing, both the enzyme and its substrates, interact to result in the production of HCN. In recent years linamarase has attracted considerable industrial and academic interest in view of its potential use in the processing of edible cyanogenic plant tissues, particularly cassava, apricots and bitter almonds and the quantification of bound cyanide in the form of cyanogenic glucosides (CNG) in plant tissues and body fluids. For example, linamarase has been

Introduction

Linamarase (/]-D-glucohydrolase, EC 3.2.1.21) along with its naturally occurring substrates, linamarain (2hydroxyisobutyro-nitrile-/]-D-glucopyranoside) andlotaustralin (2-hydroxy-2-methylbutyro-nitrile-/~-D-glucopyranoside) is found in a variety of edible plant tissues such as those of cassava (Manihot esculenta Crantz), flax (Linum ussitatissimum), white clover (Trifolium repens, L.), butter beans (Phaseolus Lunatus L.), peaches, apricots and bitter almonds. The toxicity of these plants is associated with the linamarase-catalyzed hydrolysis of these two structurally-related cyanogenic *To whom all correspondence should be addressed, e-mail: lkediobi(~/Talstar 491

492

Ikediobi et al.

used successfully in a batchwise process to detoxify fermenting cassava during 'gari' production [2,3]. In order to minimize waste associated with batchwise use of the soluble enzyme, few attempts have been made to immobilize linamarase with a view to develop a system that enabled repeated use of the enzyme [4-6]. Unfortunately, extensive data on the effects of different immobilization methods on the kinetic performance of linamarase are scanty. We report here on the immobilization, and some kinetic properties of cassava linamarase as part of our continuing effort to develop an immobilized linamarase system suitable for analytical and processing applications. Materials and methods

Chemicals Linamarin, pNP-/J-D-glucopyranoside, other pNP-glycopyransosides, 7-aminopropyltriethoxysilane, glutaraldehyde and non-porous glass beads (3 mm diameter) were purchased from Sigma Chemical Co. (St Louis, MO). All other chemicals and reagents used were of analytical grade.

Purification of linamarase Purified linamarase was prepared from cassava cortex, essentially as described in the literature [7,8]. The extent of purity of the preparation was established by analytical SDS-polyacrylamide gel electrophoresis.

mixture incubated for 1 h at 4°C. At the end of the reaction, the beads were decanted, washed with double distilled water to remove unimmobilized enzyme and then stored at below 4°C until used for kinetic studies.

Assay of linamarase Linamarase activity was assayed by the methods of Cooke et al. [7], as modified by Ikediobi et al. [8].

Kinetic studies Initial velocity studies were conducted with several different concentrations of linamarin and pNP-/3-D-glucopyranoside as substrate in the presence and absence of each of the inhibitors, glucono-l,5-1actone and imidazole. Lineweaver-Burk double reciprocal plots were prepared to obtain V ...... K,n and K~ data.

Operational stability To determine operational stability, the activity of the immobilized linamarase, was determined daily for 4 weeks under actual use conditions. Assuming that loss of enzyme activity (protein denaturation) is a first order process, a plot of log (enzyme activity remaining) vs time in days was made to yield a straight line from which the operational stability of the immobilized enzyme was estimated.

Results and discussion

Immobilization of linamarase Non-porous glass beads were cleaned with hydrofluoric acid, rinsed and then soaked in 10 M NaOH at 80°C for 1 h. Beads were rinsed with double distilled water and dried. The clean, dry beads were treated with 2% ~,-aminopropyltriethoxysilane in acetone. This was followed by reaction with 10% glutaraldehyde in 1.5 M sodium phosphate buffer, pH 6, for 15 rain. The beads were then decanted and a linamarase solution containing 14 units/ml was added to the beads and the

Contrary to current practice, the most efficient immobilization of this enzyme was achieved under conditions in which the silanization reaction was carried out in a non-aqueous medium (acetone in this case) and the cross-linking bifunctional reagent, glutaraldehyde, used at the level of 10%. Figure 1 shows the general plan of the three-step immobilization process, which consists of the silanization of the glass surface (Fig. 2), cross-linking reaction with bifunctional glutaraldehyde (Fig. 3) and finally covalent attachment of

Silanizationof glass surface

0 Gtutaraldehyde

crosslinking

~/G~

E

Covalent Attachment

of enzyme

0

Fig. 1. The general approach to covalent attachment of linamarase to glass beads: G = glutaraldehyde; E = Linamarase.

Immobilization q["linamarase

on

I

I

O I --Si-OH I O I ~Si-OH

O I

~Si-OI O

H2N(CH2)3Si(OCH2CH3)3

+

7-aminopropyltriethoxysilane

I

I

O

I

SI (CH2)3NH 2 I O I

(CH2)3NH 2

I

0

0

I

I

I

of the surface of glass beads

linamarasc to the carbonyl group of glutaraldehyde via a Schiff~ base linkage (Fig. 4). The percentage of protein and enzyme activity immobilized were detcrmined to be 92 and 86, respectively. Table 1 presents a summary of some kinetic properties of immobilized and unimmobilized linamarase. While the immobilized and unimmobilized preparations were very active with linamarin and p-nitrophenyl-fl-D-glucopyranoside as substrates, enzyme activity data for both the immobilized and soluble enzymes with such other p-NP glycoside substrates as pNP-fl-D-fucopyranoside, pNP7-D-galactopyranoside, pNP-~-D-glucopyranoside, pNP/~-D-mannopyranoside, pNP-z~-D-gentiobioside, pNP-~D-galactopyranoside and pNP-fi-D-gentiobioside were very low or insignificant. As a result, it was not necessary to obtain Km values for the immobilized enzyme with respect to these substrates. Table 2 presents the inhibition patterns obtained for the immobilized and soluble enzymes with glucono-l,5-1actone and imida-

I

I

O

O

--Si-O-

I

I

O

I

I

l

+

I

0 I

0 I

I I

o

I

I

O

(CH2)3

~Si-OI 0

ICHO

(CH2)3NH 2

zole as inhibitors. The fact the inhibition constants (KJ and the Michaelis-Menten constants (Kin) of the immobilized and unimmobilized enzymes are almost identical (Table 1) indicates that (i) both types of linamarase preparation are about equally accessible to substrates and inhibitors, (ii) immobilization may not have altered significantly (if at all) the geometry of the active site of the enzymc, and (iii) there is no diffusional limitation imposed on the substrates and inhibitots during enzyme catalysis. The competitive inhibitor, 1,5-glucono lactone, is thought to act as a transitionstate analog. This work also demonstrates that the use of a non-porous matrix in enzyme immobilization may be the solution to the vexing problem of diffusional limitation, which not only alters the kinetics of immobilized enzymes but also renders these immobilized enzymes generally less efficient than their soluble counterparts. Thc operational stability of immobilized linamarase was established to be 14 days

CHO

Si (CH2)3NH 2

O

--Si-O--Si

Fig. 3. Activation

I

O

--Si-O--SI

I

Fig. 2. Silanization

493

nrmporous glass head~

I i

o II

Si (CH2)3N----CH(CH2)30-H I O

~Si-O--SilI (CH2)3N=::=CH(CH2)31~'-H O

Glutaraldehyde

I

O

I

O

of silanized glass beads.

I i

O

1 O

I O

o

I i

o II

--si-o- sl (CH2)3N:==CH(CH2)zC-H I

o

I

ENZYME--NH 2

I

--SI--O--SI[I

(CH2)3N==CH(CH2)31C~'H

O

o

I

I

o I

I

o I

- - S l - O - Sl (CH2)3N I I O o I I - - Si--O--Si ( C H 2 ) 3 ~

I

o

I

I

o

I

Fig. 4. Cowdent linkagc of linamarase via Schifrs base.

CH(CH2)3CH

N--ENZYME

CH(CH2)3CH

N--ENZYME

b

Ikediobi et al.

494 Table 1. Some kinetic properties of linamarase enzyme

Linamarin

Km (mM) Ki (mM) Glucono- 1,5-1actone Ki (mM) Imidazole Energy of activation E. (kcal/mol)

pN P-fi-D-Glucopyranoside

Unimmobilized enzyme

Immobilized enzyme

Unimmobililzed enzyme

Immobilized enzyme

3.35 1.92 1.99 3.96

3.42 2.04 5.97 3.96

2.36 0.71 29.93 5.51

4.10 0.97 38.34 5.70

Table 2. Inhibition patterns for immobilized and soluble linamarase with glucono-l,5-1actone and imidazole as kinetic inhibitors Linamarin

pNP-fi-D-Glucopyranoside

Non-immobilized enzyme

Immobilized enzyme

Non-mobilized enzyme

Immobilized enzyme

CI CI

CI CI

CI NCI

CI NCI

Glucono- 1,5-1actone lmidazole CI = competitive inhibition. NCI = non-competitive inhibition.

at 27°C under conditions in which the enzyme was used in a fixed-bed reactor for the hydrolysis of linamarin or p-NP-/3-D-glucopyranoside. The unusual stability of the soluble (unimmobilized) preparation of this enzyme has been observed in an earlier study [9]. It is thus feasible to use immobilized linamarase to hydrolyze CNG present in industrial liquid waste from cassava or apricot processing plants prior to spectrophotometric determination and enzymatic detoxification of the free cyanide (CN) [9-12].

8.

References

9.

1. Itoh-Nashida, T., Hiraiwa, M. and Uda, Y., Purification and properties of //-D-glucosidase (Linamarase) from the butter bean Phaseolus lunatus. Journal of Biochemistry 1987, 101, 847-854. 2. Ikediobi, C. O. and Onyike, E., The use of linamarase in 'gari' production. Process Biochemistry 1982, 17, 2-5. 3. Ikediobi, C. O. and Ogundu, E. C., Production of linamarase by Aspergillus sydowii and Fusarium equiseti. Process Biochemistry 1985, 20, 99-103. 4. Ikediobi, C. O. Immobilization of Linamarase on Polyacrylamic Gels. Unpublished results, 1986. 5. Narinsigh, D. D., Jaipersad, D. and Chang-Yen, I., Immobilization of Linamarase and its use in the

6.

7.

10. 11.

determination of bound cyanide in cassava using flow injection analysis. Analytical Biochemistry 1988, 172, 89-95. Yeoh, H. H. and Tan, C. K. C., Determination of linamarin in cassava using enzyme-sensitized microcentrifuge tubes. Journal of Science, Food and Agriculture 1994, 66, 31-33. Cooke, R. D., Blake, G. G. and Battershill, L., Purification of cassava linamarase. Phytochemistry 1978, 17, 381-383. Ikediobi, C. O., Ibrahim, S. and Ogdonna, A. I., Linamarase from Fusarium equiseti. Applied Microbiology and Biotechnology 1987, 25, 327-333. Ikediobi, C. O., Onyia, G. O. C. and Eluwah, C. E., A rapid and inexpensive enzymatic assay for total cyanide in cassava (Manihot esculenta Crantz) and cassava products. Agricultural and Biological Chemistry 1980, 44, 2803-2809. lkediobi, C. O., Olugboji, O. and Okoh, P. N., Cyanide profile of component parts of sprouted sorghum. Food Chemistry 1988, 27, 167-176. Ikediobi, C. O., Latinwo, L. M., and Ling, W.,

Spectrophotometric Quantification of Inorganic Cyanide in Industrial Wastewater. American Environmental Laboratory, 1997, 9, 18-23. 12. Ling, W., Degradation of Inorganic Cyanide by Microbial Enzyme(s). M.Sc. Thesis, Florida Agricultural and Mechanical University, Tallahassee, Florida, USA. 1996.